Therapeutics based upon mammalian viruses have tremendous potential in the context of genetic disease, cancer therapy and vaccine development. Adenovirus (Ad) plays a leading role in these applications as it is delivers genetic payloads extremely efficiently, has large transgene capacity, is able to infect both dividing and quiescent cells, and poses little risk for insertional mutagenesis. As adenovirus based therapeutics mature it has become obvious that understanding and manipulating biodistribution plays a critical role in increasing efficacy. Our long-term goal is the development of techniques that allow the straightforward and selective modification of mammalian viruses with imaging reagents. The objective of this proposal is to develop highly specific chemical approaches to adenovirus modification with PET reagents. Our central hypothesis is that chemoselective reactions will allow flexible modification with PET reagents without significant impact on either biodistribution or viability. The specificity required for this approach cannot be accessed with traditional protein chemistry. We have developed a two-step labeling protocol requiring the metabolic placement of unnatural amino acids and/or sugars followed by exquisitely selective chemical modification using either click chemistry or the Staudinger Ligation. The rationale for the proposed research is once a facile and flexible imaging platform has been developed it will accelerate translational studies with Ad and, as it is expected be broadly applicable, other viruses.